Dopamine mediates the reward for such adaptive behaviors as eating and sexual activity. Drugs of abuse circumvent the requirement for normal stimulation by releasing dopamine directly, and produce long-term changes in the release of dopamine that presumably contribute to tolerance, physical dependence, and craving. Drug abuse thus provides a paradigm for synaptic plasticity in the reward pathway. The long-term goal of this program is to understand how neurotransmitter release is regulated and how changes in release contribute to drug abuse and packaging of monoamines and acetylcholine (ACh) limits their release, we will focus on the proteins that transport these and other classical transmitters into neurosecretory vesicles. In addition, we have found that the neuronal vesicular amine transporter (VMAT2) and the vesicular ACh transporter (VAChT) undergo phosphorylation. However, the phosphorylation of these proteins appears to influence their subcellular localization rather than their intrinsic transport activity. Indeed, localization of the transporters to multiple populations of neurosecretory vesicles confers the potential for multiple modes of release. In PC12 cells, VMAT localizes predominantly, to large dense core vesicles (LDCVs) and phosphorylation of an acidic patch at the C-terminus appears to redistribute VMAT2 out of LDCVs and into a perinuclear location. In contrast, the closely related VAChT appears predominantly on synaptic vesicles (SVs). Phosphorylation upstream of a previously characterized dileucine motif mimics a glutamate present at the equivalent position in VMAT2 and appears to redistribute VAChT, like VMAT2, into LDCVs. We will now: 1) Determine how VMAT2 phosphorylation influences localization to LDCVs. 2) Determine how the dileucine motif influences trafficking of the transporters. 3) Extend the analysis of membrane trafficking to primary neuronal culture. 4) Characterize the cytosolic machinery involved in trafficking of the transporters. The results will provide some of the first information about the sorting of any membrane protein to specialized secretory vesicles. In the process, it will also indicate ways to manipulate the sites of storage and release in vivo by genetic manipulation in mice and hence determine the physiological role of release from multiple vesicle populations as well as its regulation in drug abuse and other behaviors.